Poxvirus Entry, Assembly and Egress

National Institute Of Allergy And Infectious Diseases

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Abstract

There are two major types of infectious vaccinia virus particles: mature virions (MVs) and enveloped virions (EVs). The MVs, which consist of a DNA-protein core surrounded by a lipoprotein membrane, are assembled in cytoplasmic viral factories and contain about 80 polypeptides. A subpopulation of MVs is wrapped by modified trans-Golgi or endosomal cisterna containing additional viral membrane proteins, transported along microtubules to the cell periphery, and exits the cell through the plasma membrane. The EVs are essentially MVs with an additional membrane that is disrupted prior to fusion of the MV with the cell during entry. The first step in virus replication is entry. The mechanisms used by poxviruses are complicated by the existence of two distinct infectious forms with different external membranes. Another complicating factor is the ability of vaccinia virus to enter cells through the plasma membrane and by endocytosis. We have identified a complex comprised of at least 11 proteins that are conserved in all poxviruses and required for entry of both infectious forms of virus. These proteins are conserved in all members of the poxvirus family, suggesting a common entry mechanism. Poxviruses also encode proteins that localize in cell membranes and negatively regulate superinfection and syncytium formation. The vaccinia virus (VACV) A56/K2 fusion regulatory complex associates with the G9/A16 EFC subcomplex, but functional support for the importance of this interaction was lacking. Two studies completed during the past year suggest a role for the O3 protein in stabilizing the entry fusion complex. The vaccinia virus O3 protein is comprised of only 35 amino acids of which 20 amino acids form a putative transmembrane domain. Deletion of the O3 gene greatly impairs virus entry and results in a small plaque phenotype. Experimental evolution was carried out by blindly passaging the deletion mutant. Large plaque variants arose spontaneously, and genome sequencing of individual cloned viruses revealed mutations in predicted transmembrane domains of three open reading frames encoding proteins with roles in entry. Further analyses indicated that the adaptive mutants also had higher infectivity, entered cells more rapidly and increased EFC assembly, which partially compensated for the loss of O3. These results suggest that one role of O3 is to interact with the transmembrane domains of other EFC proteins. In another approach, we adapted the tripartite split green fluorescent protein (GFP) complementation system to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP beta-strands. To achieve fluorescence, two additional 20-amino acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus expressing GFP1-9 and plasmids that express individual EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy and by flow cytometry. Previous EFC protein interactions were confirmed, and new ones discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. Together, the two studies suggest that one role of O3 is to interact with the hydrophobic domains of the EFC proteins to stabilize their association.